Cholesterol is an essential component of cellular membranes, a regulator of membrane protein function, and an obligate metabolic precursor. Exogenous cholesterol is acquired principally through receptor-mediated endocytosis of cholesteryl ester-laden low-density lipoproteins and trafficked to the lysosome. Lysosomal acid lipase liberates unesterified cholesterol, which is transferred by the Niemann-Pick C1 and C2 (NPC1, NPC2) proteins to the limiting lysosomal membrane and subsequently to other cellular organelles. Cholesterol exiting the lysosome is distributed to multiple compartments ? including the plasma membrane, ER, Golgi, mitochondria and peroxisomes ? and serves to regulate key cholesterol homeostatic responses. While several proteins have been implicated in post-lysosomal cholesterol trafficking, the components and relative contribution of each pathway and specific trafficking itinerary of the lipoprotein-derived cholesterol are poorly understood. This represents an important gap in our knowledge and an area in which advances have the potential to identify new therapeutic targets for treating both atherosclerotic and neurodegenerative diseases. We have generated novel cholesterol probes that can substitute for cholesterol and allow for the selective capture of cholesterol interacting proteins for unbiased identification by mass spectrometry. These probes provide a powerful new tool to study previously elusive post-lysosomal cholesterol trafficking pathways. We hypothesize the specific itinerary and metabolic fate of lipoprotein cholesterol involves both NPC1-dependent and -independent distribution pathways that can be identified based on unique cholesterol cargo interactomes. Elucidation of these protein networks through quantitative proteomics will identify major nodes through which lipoprotein cholesterol traffics and novel therapeutic targets..